This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. This was a subcontract with Pacific Northwest National Laboratories to characterize the comprehensive proteome of defined orthopoxvirus particles of known pathogenicity. The orthopoxviruses that we focused on were wild-type nonpathogenic vaccinia virus (VV;Western Reserve strain) and pathogenic human monkeypox virus (MPV;Zaire strain). In this objective, the protein components of the extracellular enveloped virus (EEV) and intracellular mature virus (IMV) were determined for pathogenic MPV, and compared to that of VV. Having evaluated and compared virus particles, we shifted our focus towards alterations to the host cell following infection. Here we compared the intracellular and extracellular proteome of VV and MPV infected HeLa cells with the goal to identify novel viral and cellular proteins that are synthesized in response to pathogenic orthopoxvirus infection. From these studies, we found that specific viral encoded proteins reported to be secreted from VV-infected cells were also secreted from MPV-infected cells. Interestingly, we also identified another viral encoded protein that has no known reported function abundantly secreted in the media. This finding is not surprising, as there are a number of annotated open reading frames with no known homologue. As such, the detection of this viral encoded protein in the supernatant of infected cells suggest the protein has a yet to be identified function that can potentially impact the function of surrounding cells. Additionally, we initiated proteomic analysis on bronchio-alveolar samples derived from animals experimentally infected with MPV to identify viral encoded proteins that facilitate MPV-associated pneumonia.